Article Figures & Data
Figures
- Figure 1
Cholinergic stimulation by ACh boosts spontaneous action potential firing in Hcrt+ neurons. A1 , Sketch of a brain slice showing Hcrt+ neurons (green cells in the red box) residing in the hypothalamus. A2 , Morphology of two Hcrt+ neurons shown in the fluorescent channel. A3 , Differential Interference Contrast video-microscopy showing the experimental paradigm of pressure application (puff) of drug onto the soma and proximal processes, while keeping the patch onto the neuron. Scale bar, 10 µm. B1 , Mechanical interference (puff of bath solution) frequently produces a temporary depression of spontaneous firing. B2 , In the presence of atropine (4 µM), application of ACh (1 mM) boosts action potential firing for tens of seconds. B3 , The same trace as in B2 , except on a different scale and filtered with Gaussian low pass 5 Hz. B4 , Injection of +10 pA of current into the Hcrt+ neuron notably increases the firing frequency. Red lines represent the timing and duration of ACh application or current injection. C, Mechanical interference does not affect firing (n = 4/11 cells), or results in a temporary depression of firing (n = 7/11 cells). D, Differential responses to the puff of ACh. In 7 out of 20 cells, firing was enhanced by ACh, 4 out of 20 cells were not affected, while 9 out of 20 cells were inhibited. E, With DHβE in the bath, ACh did not increase firing of any Hcrt+ neurons tested (n = 11/11 cells). F, With MLA in the bath, a puff of ACh boosted firing in 2 of 16 cells, had no effect in 9 of 16 cells, and decreased firing in 5 of 16 cells. The gray bar indicates the time duration of ACSF application; yellow bars indicate the time duration of ACh application. C−F, All experiments were conducted in the presence of atropine. G, The responses of Hcrt+ neurons to ACh in absence of atropine. In 8 out of 14 cells, firing was enhanced by ACh, in 1 out of 14 cells firing was unaffected, while in 5 out of 14 cells firing was inhibited.
- Figure 2
The effects of α4β2 receptor antagonist, DHβE, and α7* receptor antagonist, MLA, on the action potential firing of Hcrt+ neurons. Top, Change of firing rate (action potentials/5 s) before and after bath application of DHβE (red) and MLA (blue). Bottom, A representative cell reduced in firing rate upon bath application of DHβE, and partially recovered over time.
- Figure 3
Nicotinic stimulation induced variable inward currents in a third of Hcrt+ neurons. A1 , A small, desensitizing current evoked by ACh (1 mM) puff, in the presence of atropine (4 µM). A2 , Large, repetitive currents evoked by ACh (1 mM) puff every 60 s. B, In 32 out of 92 Hcrt+ neurons, an ACh (1 mM) puff evoked inward currents of variable size. C, In 9 out of 29 Hcrt+ neurons, a nicotine (Nic; 100 µM) puff evoked inward currents of variable size. D, E, Mean sizes of currents evoked by puffing ACh (1 mM; B) or nicotine (100 µM; C), respectively, onto Hcrt+ neurons.
- Figure 4
ACh suppresses glutamatergic spontaneous mEPSCs onto Hcrt+ neurons in the presence of atropine. A, Histograms of mEPSC instantaneous frequency show the time course of the effect of ACh on glutamatergic mEPSCs from synapses impinging onto Hcrt+ neurons. Bars above the graph indicate the time point of the ACh (1 mM) puff. The original recordings indicated by “Baseline”, “Ach”, and “Wash” are displayed in B1 , B2 , and B3 , respectively. Inset shows the full trace of an example mEPSC event. C, Cumulative probability plot of the mEPSC amplitude at baseline and upon ACh application from six cells. Inset shows the mean value of the mEPSC amplitude at three time periods: baseline, under the influence of ACh, and washout. **p < 0.01. D, Interevent intervals of mEPSCs at baseline and upon ACh application from six cells (same cells as in C). Inset shows the mean of instantaneous frequency of the mEPSC at three periods of time: baseline, under the influence of ACh, and after washout.
- Figure 5
Effects of nicotine exposure on glutamatergic mEPSCs onto Hcrt+ neurons. A, Histograms of mEPSC instantaneous frequency showing the effect of nicotine (Nic) exposure (100 µM) on mEPSCs onto Hcrt+ neurons. The arrow indicates the time of nicotine application. Depending on the response pattern, two periods of time (baseline and Nic) were used for analysis. B, C, Mean value of amplitude (B) and instantaneous frequency (C) at baseline and under the influence of nicotine. n = 4 of 11 cells. **p < 0.01. D, Histograms show another representative response to nicotine (1 µM) on mEPSCs onto an Hcrt+ neuron. Three periods of time (baseline, T-1, and T-2) were used for analysis. E, F, Mean value of amplitude (E) and instantaneous frequency (F) of mEPSCs during baseline recording and at two periods after nicotine exposure. n = 4 of 11 cells.
- Figure 6
Nicotinic AChR antagonists suppressed glutamatergic mEPSCs onto Hcrt+ neurons. A, Mecamylamine, a nonselective nAChR antagonist, strongly suppressed mEPSCs onto Hcrt+ neurons. The bar above the graph indicates the duration of MEC (10 µM) exposure. Miniature EPSCs from two periods of time (Baseline and MEC) were used for analysis. B, Cumulative probability plot of the mEPSC amplitude at baseline and during MEC exposure was made from the recordings of four cells. Inset shows the mean values of the mEPSC amplitude at baseline and following MEC exposure. **p < 0.01. C, Interevent intervals of mEPSCs at baseline and upon MEC application from four cells (same cells as in C). Inset shows the mean instantaneous frequency of the mEPSCs at baseline and following MEC exposure. D, E, DHβE, an antagonist of heteromeric nAChRs (particularly α4β2 nAChRs), moderately suppressed the amplitude (D) and instantaneous frequency (E) of mEPSCs onto Hcrt+ neurons. n = 7 cells. F, G, MLA, a relatively selective α7 nAChR antagonist, partially suppressed mEPSC amplitude (F) and instantaneous frequency (G). n = 8 cells.
- Figure 7
Inhibiting calcium release from internal stores occludes the effect of ACh on mEPSCs onto Hcrt+ neurons. A, B, mEPSCs recorded at baseline (A) and after 30 min incubation with the ryanodine receptor antagonist dantrolene (50 μM) and the IP3 receptor antagonist (−)-Xestospongin C (5 μM; B). C, D, Mean values of amplitude (C) and instantaneous frequency (D) of mEPSCs at baseline, after 30 min incubation with dantrolene (Dan) and XeC and upon subsequent ACh puff in the continued presence of dantrolene and XeC. n = 3 cells. Both amplitude and instantaneous frequency were significantly decreased by dantrolene+XeC, but there was no significant difference between mEPSC recordings in the presence of dantrolene and XeC and upon subsequent ACh puffs in the continued presence of these inhibitors. **p < 0.01; NS, not significant (p > 0.05).
- Figure 8
ACh increased action potential (AP) firing or had no effect. In the same cells, however, ACh decreased mEPSC occurrence despite of its effect on action potential firing. A, A Hcrt+ cell increased action potential firing rate in response to ACh, in presence of atropine. B, In the same cell, a puff of ACh decreased mEPSC frequency. Yellow bars indicate the time duration of ACh application. C, Another Hcrt+ cell did not change action potential firing rate upon ACh puff. D, In the same cell, a puff of ACh decreased mEPSC frequency.
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